Method of hatching avian eggs

ABSTRACT

A method of hatching eggs of avian species, especially chickens, which comprises carrying out at least one cycle of steps (a) and (c) as follows: (a) incubating the eggs in a first, baseline ambient environment which is normal for hatchery incubation of the eggs of the avian species in the prevailing climatic conditions and then during a sex-sensitive time window of embryonic development, (b) altering the ambient environment to shifted conditions for a period of time effective to bias the normal phenotypic sex ratio of the embryos, without significant adverse effect on the average mortality rate, and (c) thereafter restoring the incubation conditions to or towards normal and allowing the eggs to hatch. Preferably the ambient environment comprises a temperature of 37.5°-38° C. and the shifted conditions comprise reduced temperature, especially a temperature maintained at about 22° C. for a period of from 18 to 42 hours. The method may be used to produce birds which are chromosomally male (ZZ) but phenotypically female or chromosomally female (ZW) but phenotypically male.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the hatching of eggs of avian species,especially of poultry birds, and to the hatchlings thus obtained, adultbirds grown from the hatchlings and subsequent generations of birds bredfrom said adult birds.

2. Description of Related Art

Sex determination in avian species, including domestic poultry(chickens, turkeys, pheasants etc) is determined by genetic mechanisms.Female birds are heterogametic, with ZW chromosomes, whereas males arehomogametic, with ZZ chromosomes (this is the reverse of the situationin mammals, where males (XY) are the heterogametic sex and females (XX)are the homogametic sex. It is assumed that there are genes on eitherthe Z or the W chromosome, responsible for sex determination in birds.However, it is unclear whether the genes which regulate male or femaledevelopment are present on the Z chromosome, the W chromosome or on somecombination of these (e.g. an activator on the Z chromosome and aninhibitor on the W chromosome). What is clear however, is that in birds,as in mammals, sex is apparently determined at the time of fertilisation(Sittman 1984, and references therein).

By contrast, in some reptiles, the temperature of egg incubationdetermines the sex of the offspring; there being no heteromorphic sexchromosomes in either males or females (see Deeming and Ferguson 1988for a review),

It has therefore been supposed that there are two fundamental types ofsexual determination in vertebrates: genetic (or chromosomal) sexdetermination as in birds and mammals, whereby the sex of the offspringis determined at the time of fertilisation and environmental (ortemperature) determination in reptiles where sex is determined afterfertilisation, as a result of interaction with some environmental agent.

There have been few previous investigations of the effects oftemperature on sex ratio in birds, probably because deviations from thenormal temperature in birds are usually lethal to the embryo (Bennettand Dawson 1979, Tazawa and Rahn 1986). In the domestic chicken,deviations from the preferred incubation temperature of 37.5° to 38° C.during incubation causes significant embryonic mortality. This situationin birds if very different from that In reptiles whose embryos aretolerant to a fairly wide range (often 5° C. or more) of incubationtemperatures without any significant effect on embryonic mortality.

However, Shubina et al. (1972) have reported that a change in incubationtemperature of chicken eggs can alter the sex ratio between male andfemale embryos. The paper begins by referring to an earlier paper whichis said to have shown that a brief increase in incubation temperature inthe period preceding or concurrent with gonadal differentiation ofchicken embryos leads to the formation of primarily female individuals.There follows a description of experiments apparently showing thatreducing the temperature of incubation after 72 hours from 37.5° C. to22° C. for 7 or 8 hours biases the sex ratio towards males, namely to a1.5:1 or 1.43:1, respectively, ratio of males to females. These figuresare not encouraging because mortality of the embryos was 9.1 and 10.5%respectively, compared with 6.1% in the control group: see Table 1.However, all figures in Table 1 must be treated with caution, since itis unclear how to reconcile the percentage mortalities with the totalnumber of embryos examined and the total number of eggs used. Also, oneset of experiments seems to have been omitted from the results shown inTable 1. Thirdly, the paper reports in Table 2 a previous experiment inwhich the incubation temperature is increased to 41° C., yet themortalities reported are only 7-12% and the bias is in favour of males.

SUMMARY OF THE INVENTION

The present invention provides a method of hatching eggs of avianspecies, especially of chickens, which comprises carrying out at leastone cycle of steps (a) to (c) as follows:

a) incubating the eggs in a first, baseline ambient environment which isnormal for hatchery incubation of the eggs of the avian species in theprevailing climatic conditions and then during a sex-sensitive timewindow of embryonic development, (b) altering the ambient environment toshifted conditions for a period of time effective to bias the normalphenotypic sex ratio of the embryos, without significant adverse effecton the average mortality rate, and (c) thereafter restoring theincubation conditions to or towards normal, and allowing the eggs tohatch.

The eggs will normally be incubated at 37.5° C.-38° C. The alteration ofthe ambient environment to shifted conditions normally takes the form ofa reduction in temperature of incubation, especially to either about 22°C. or to about 36° C., whereby the sex ratio of hatchlings is biased infavour of males or females respectively.

The terminology "without significant adverse effect on mortality rate"implies that it is readily possible to find conditions in which themortality is below 9% as determined on embryos at day 10 of incubation.More preferably, mortality is below that of control eggs incubated at aconstant 38° C. These conditions differ from those of Shubina et al.supra, especially in respect of the period of time during which thetemperature is shifted, also referred to herein as "pulse time".

To carry out the method an incubator should be adapted to carry out acycle of steps (a) to (c) preferably by the provision of associatedprogrammable control means for varying the ambient conditions referredto, especially temperature. point data, places in the incubator whereeggs could be placed to ensure uniform incubation temperature weredetermined. In all subsequent experiments, eggs were placed in theseuniform temperature zones within the incubator. Further, it was observedthat when the incubator lid was opened for more than 5 minutes, then thetemperature in the incubator rose to above that at which it was set.This was obviously a function of the thermostat, activating the heaterin the incubator to warm it up again. It was determined in earlyexperiments that these temperature spikes were detrimental to the eggsand complicated the experiments. Accordingly, to avoid all temperaturespikes, all eggs were placed in the incubators with the temperaturecontroller set at 36° C., which prevents temperature spiking above 38°C. Once all the eggs were placed in the incubator and the lid firmlysecured, the temperature controller was then raised to 38° C. It tookthe incubator approximately 60 minutes to equilibrate after placing theeggs in it in this fashion. Therefore incubation was reckoned to beginafter this 60 minute period. All references herein to incubation periodsare to be construed accordingly.

EGGS AND METHODS OF DETERMINING THE SEX OF EMBRYOS

The chicken eggs were from Rhode Island Red hens and were purchased froma commercial supplier.

In preliminary investigations, embryos were recovered on days 3-12inclusive, their abdomens opened and the animals sexed according to therelative size of the gonads. The gonads were also removed forhistological sectioning to confirm their sex. The earliest time pointfor reliable sexing of the embryos was day 7 of incubation. Allembryonic sexing reported In this study was performed on embryonic days9,10,12,17, and 20. The embryonic day chosen did not affect the results.

Controls

In all experiments a control batch of eggs was incubated at a constant38° C. throughout. This should produce a roughly 50/50 sex ratio,although in the Isabrown species, there is consistently a higherpercentage of females, compared to males (sex ratio approximately 1:0.9). If these control eggs showed significant mortality, i.e. above10%, all experimental results were disregarded, and none of these arereported. This has happened on several occasions, and may be due tostress in the breeding stock, change in diet or other unrelated factors.

Collection of eggs from the hatchery

Eggs were collected from the birds shortly after laying. They werestored at the hatchery and transported at ambient temperature toManchester University, where they were kept at ambient temperature for amaximum of 24 hours before commencing incubation. There is normally nodevelopment during this period. This is the normal poultry industryregime. If eggs are incubated immediately after laying, many embryosdie.

Temperature shifting (pulsing)

At various times during incubation (as described in the Tables whichfollow) eggs were pulsed with either a higher or lower temperature for avariable pulse duration (as described in the Tables). Thus, for a shifton embryonic day 3, eggs were incubated for 72 hours (plus or minus 30minutes) before the incubator was adjusted to pulse them, for example,at 22° C. for 48 hours (in the case of the two day pulse). Pulsing ofthe eggs was achieved by quickly placing the incubators in a cold room(4° C.) with the incubator lid ajar about 4 cm. This allowed for rapidcooling of the eggs within the incubator, which then rapidlyequilibrated to its newly set temperature. Thus, for example, theincubator reached 22° C. within 15 minutes under this regime. The timeat which the pulse temperature was reached was taken as the 0 time pointfor measuring the length of the pulse. In each experiment, 1 or 2 eggswere removed from the incubator at the commencement of the pulse time,opened and the embryo stage according to Hamburger and Hamilton criteriadetermined. Each incubator was carefully adjusted and monitored duringthe pulse period to maintain constant temperatures throughout the pulse.

Restoration of normal incubation condition

At the end of the pulse period, the incubator was then adjusted to bringthe temperature back up to 38° C. This took no more than 30 minutesbetween setting the incubator controls and the eggs reaching their newtemperature. Equally, there was no spiking of the incubator in terms oftemperatures exceeding 38° C. The incubation of the eggs was continuedat 38° C. until the time of termination (normally embryonic day 10 orday 17). During this time period, the incubator temperatures wereconstantly monitored with the platinum resistance thermometer probes,calibrated against the National Standard thermometer.

Examination of embryos (sexing and determining mortality)

At the end or the incubation period, the eggs were killed and opened andthe embryos removed and staged according to the Hamburger-Hamiltonmethod. All infertile eggs were noted and discarded from any furthercalculations. Eggs containing dead embryos were noted, and if possible,the sex of the embryo was determined (this is usually not possible). Ofthe remaining living embryos, these were opened and the embryo sexed bythe macroscopic appearance of the gonads. In cases of doubt and in anycase in one in five embryos, the gonads were removed and processed forhistological sectioning. Sex was then diagnosed on the basis of gonadaldifferentiation into either testis or ovaries. The percentages of male,female and dead embryos in the Tables were derived in this way.

Genotyping

The heads of each of the embryos were removed and frozen at -70° C. Thistissue was used to extract genomic DNA for slot blot hybridisation witha chicken W chromosome probe (Tone et al., 1982, Kodama et al., 1987,Saitoh et al., 1992, Saitoh and Mizuno 1992). This probe is a specificmarker for the W chromosome and was used to confirm the genetic sex ofthe embryos. Slot blot analysis was only performed In experiments wherethere was a significant deviation of the sex ratio observed uponmacroscopic and histological examination of the gonads. It served toconfirm that the macroscopic phenotypic sex of the embryo differed fromits genotypic sex on the basis of the W-chromosome probe.

Based on the macroscopic, histological and DNA analysis of sexdetermination, there was no difference in the accuracy of sexing embryosat any of the embryonic days/stages used. Moreover, in repeatexperiments of the same temperature pulse conditions, there was nosignificant difference in the percentage of shift of the sex ratiodependent upon the embryonic age of sex determination. Confidence cantherefore be placed in the accuracy and reproducibility of these sexingmethods.

RESULTS

The data in Tables 1-3 clearly show that sex reversal occurs reliablyand reproducibly under a certain set of conditions. Under other sets ofconditions, sex reversal is not predictable, i.e. it happens some times,but not others, and under a wide range of conditions, there are noeffects. This allows one to define a matrix of optimum conditions foralterations in the sex ratio. This can most easily be described by:timing of onset of the pulse, duration of the pulse, and temperature atwhich the eggs are pulsed.

Time of onset of the pulse in days and Hamburger Hamilton stages

The sensitive time window of development where a shift in temperaturesignificantly affects the sex ratio, is between embryonic days 2-4 withthe optimum on embryonic day 3. The term "on embryonic day n", n being aspecified integer, as used herein, signifies that the pulsing took placewhen the eggs had been incubated for 24 n hours, plus or minus 30minutes. Shifting the incubation temperature on or after embryonic day 5has no effect on the sex ratio, but adversely effects the embryonicmortality. Experimental analysis was therefore concentrated on embryonicday 3.

An interesting early observation was that the stage of the embryo at thecommencement of the pulse was very important. This meant that eggs whichhad been stored for too long before incubation began, gave variableresults. This is illustrated clearly in Table 2, where a number ofpulses on embryonic day 3 for similar periods of time and pulsing tosimilar temperatures gave unpredictable results. The reason is that theeggs used in the experiments in Table 2, varied in the length of storagebetween the time of egg laying and the commencement of incubation. Thismeans that embryos were at different stages of development whencommencing the time of the pulse. It was determined that it is importantfor optimal results to commence temperature pulsing atHamburger-Hamilton stage 18/19, which is normally on embryonic day 3.The effects of pulsing on these days/stages are reproducible and areillustrated in Table 3. In practice, this means that eggs are kept forapproximately 1 day (24-36 hours) between laying and the commencement ofincubation. If eggs are stored for longer periods of time, it isimportant to titre the commencement of the temperature pulse, so that itbegins at Hamburger-Hamilton stage 18/19.

Pulsing temperature to which the eggs are shifted

Optimum skews in the sex ratio towards an increase in males wereobserved when eggs were shifted on embryonic day 3 from their normalincubation temperature of about 38° C. to a temperature of about 22° C.(Tables 1 and 3). This produced at best a sex ratio of approximately 1.6males for every female with a very low mortality figure (usually in theregion of 5% which is the control level). Also, a shift to about 36° C.on embryonic day 3 reliably biased the sex ratio towards females, againwith low mortality.

Shifting to other temperatures either has an unreliable effect on thesex ratio or causes substantial mortality of the eggs (Table 1).

Duration of the temperature pulse

Skews in the sex ratio were obtained with temperature pulses of 22° C.commencing on embryonic day 3 for variable periods between 8 and 48hours. In general, long temperature pulses, i.e. exceeding 36 hours,usually produce increased mortality. Contrariwise, short temperaturepulses have less reproducible effects. The optimum duration of the pulseseems to lie at around between 24 and 36 hours (Tables 1 and 3), but theresults at shorter and longer pulse lengths indicate some latitude inthese figures, say to a wider preferred range of 18 to 42 hours.

For temperature pulses at 36° C., preliminary experiments, reported inTable 1 of the priority application, carried out under less preciselydefined conditions than given above, indicated that a pulse length of 2to 4 days was particularly suitable and Table 1 below confirms. Again,there is some latitude in the lower figure, indicating a wider preferredrange of 18 to 96 hours.

Age of the laying flock

To investigate the possible effects of flock age on the nature of thesex reversal obtained with temperature pulses during incubation, theoptimum sex reversing regime i.e., a temperature pulse of 22° C.commencing on embryonic day 3, Hamburger-Hamilton stage 18/19 forvarying periods of time, (Table 3), was used. Table 3 shows that theeggs of older parents show more of a sex skew than that of youngerparents.

2. EFFECTS OF INCUBATION TEMPERATURE PULSING ON HATCHABILITY

To investigate whether the temperature pulsing regime adversely affectedembryonic development and caused high late embryonic mortality orproblems in hatching, the following experiment was established.

MATERIALS AND METHODS

400 eggs of the Isabrown auto-sexing strain were collected from the sameflock of hens on Apr. 20, 1993. In the Isabrown strain, sex is closelyrelated to feather colour, and hince, it is possible to sex the birds athatching by looking at their feathers as an indicator of the genotypicsex. There is a less than 1% error claimed by the suppliers of theseeggs in this feather sexing method. The Isabrown auto-sexing chickensare bred from pure parental lines. On hatching, the females are brown,later turning red, with white under-feathers. The males are yellow,later turning pure white. This auto-sexing characteristic is seen asearly as embryonic day 12, when the feathers are being formed. In theembryo, the male feathers are transparent, whereas the female feathersare reddish brown. The percentage error in determining sex by feather inthe embryo, compared to macroscopic inspection of the gonads, wasdetermined to be less than 0.01%. The eggs were controlled in terms ofparent of origin, age of the flock and age after egg laying. The eggswere placed in incubators on Apr. 21, 1993, as described previously. Thepulsing regimes consisted of the following.

On embryonic day 3, i.e. at 72 hours (plus or minus 30 minutes) from thebeginning of incubation, 100 eggs were pulsed at 22° C. for 24 hours and300 eggs were pulsed at 36° C. for 48 hours.

On May 7, 1993, all eggs were removed from the incubators at theUniversity of Manchester and candled. Candling allowed one todistinguish between fertile developing eggs, dead eggs or infertileeggs. At this stage, the dead or infertile eggs were removed anddiscarded. In the 36° C. group, 56 of the 300 eggs were infertile andnone were dead, meaning that for the rest of the experiment, there wasan effective starting group of 244 eggs. In the 22° C. shift group, 18eggs were infertile and none were dead, i.e., the experiment startedeffectively with 82 eggs. Following the discard of these infertile eggs,the viable eggs were transported immediately to hatching incubators atthe University of Liverpool Veterinary School, a distance of about 25miles (45 km.). They were placed in these hatching incubators, so as todistinguish between the two treatment groups, i.e., 36° C. and 22° C.treatments, but all these hatching incubators were set at 38° C.Separation of the eggs in the treatment groups allowed for subsequentanalysis on hatching.

The eggs hatched on May 13, 1993. The phenotypic sex of chickens on theday of hatching was determined by examining the macroscopic appearanceof the vent of the bird. This is a common procedure in poultry practice,and is the basis for discarding male or female chickens under currentpoultry practice conditions. An experienced poultry vent sexervent-sexed all the birds from these experiments on the day of hatching.He knew neither about the auto-sexing characteristics of the feathers ofthe Isabrown strain of birds, nor the details of the experimentaltreatment, i.e. sexing at hatching was done "blind". All birds were thenindividually banded, so that they could be subsequently identified andtraced to one of the experimental groups.

Results Mortality

In the 36° C. treatment group, (starting with 244 eggs), 37 eggs did nothatch or died at the time of hatching. Of these, most of the birds diedtrying to get out of the shell. In order to emulate practice in thepoultry industry, no attempt was made to assist the birds out of theshell. One chick died on the first day after hatching and four chicksdied within the first two weeks of hatching, due to disease. So, in the36° C. group, out of the total of 244 viable eggs, 85% hatched and 15%did not hatch.

In the 22° C. treatment group, of the 82 viable eggs in the experiment,13 birds did not hatch, i.e. died on or before the time of hatching.Like the 36° C. group, the vast majority of these died on the day ofhatching from a failure to get out of the shell. No chicks died in thefirst week. This means that out of a total of 82 viable eggs in the 22°C. pulse group, 84% hatched and 16% did not hatch.

The mortality figures in both groups are almost identical. This suggeststhat the mortality may have more to do with the conditions of theexperiment, i.e. transporting the eggs to different incubators shortlyafter embryonic day 16, than to the pulsing regime. In any case, themaximum mortality as assayed at the time of hatching is likely to bearound 15%.

Effects on sex at the time of hatching

The data are presented in Table 4 which documents genotypic sex, i.e.feather colour and phenotypic sex, i.e. vent sex in the varioustreatment groups. (The vent sexing is analogous to examination of thegonads of embryos).

In the 36° C. treatment group, in the 105 birds whose genotypic sex wasmale, there was complete concordance with the phenotypic sex. In the 101birds whose genotypic sex was female, one bird was phenotypically male.This bird had a feather pattern which was intermediate between that ofmale and female. This is unlikely to be due to a temperature pulsingeffect. The conclusion therefore is that 36° C. treatment had no effectin terms of sex reversal as assayed on the day of hatching. It can beregarded as a control experimental group for the purposes of sexreversal determination.

In the 22° C. group, of the 30 birds whose genotypic sex (feathercolour) was male, there was again complete concordance between genotypeand phenotype. However, of the 39 birds whose genotypic sex by feathercolour was female, only 35 had the phenotypic sex of female, asdetermined by vent sexing. The other four had the phenotypic sex of maleand were, therefore, "sex reversed", i.e. their genotype was female, buttheir phenotype male.

The sex reversed birds were reared under conventional poultry raisingconditions for five months and they appeared to grow satisfactorily andmaintain their phenotypic sex. Unfortunately, the birds were then killedby the sponsor who had agreed to pay for feeding them and this actionoccurred without any warning to the inventor or patent applicant.Nevertheless, the experiment clearly demonstrated that the temperaturepulsing regime does not produce a significant mortality in terms ofhatching, that adult birds whose phenotypic sex differs from theirgenotypic sex were produced and that these birds can develop and grownormally.

Implications

The unexpected finding that manipulation of incubation temperature indomestic chickens can influence the sex ratio, without the highmortalities reported by Shubina et al., may be important in a number ofways. First, it will be possible to skew the sex ratio towards eitherfemales for egg laying species or males for species required for maximalgrowth. Second, it is possible to generate by this invention, birdswhose phenotypic sex is different from their genotypic sex, e.g. ZWmales. When such birds breed, the new stock would produce a skewed sexratio in subsequent generations, even if all the eggs were incubated at38° C. This may be very important for commercial businesses which supplystock into the poultry industry. Thus, the invention includes a methodof producing birds of an avian species which are chromosomally male (ZZ)but phenotypically female or chromosomally female (ZW) butphenotypically male, which comprises hatching eggs by a method of theinvention, above defined, to bias the phenotypic sex ratio, detectingthe chromosome type of the hatchlings and selecting hatchlings which arechromosomally male but phenotypically female or vice versa. It furtherincludes rearing the hatchlings to an adult age at which they arecapable of breeding. Still further, it includes allowing thephenotypically female or male birds (chromosomally ZZ or ZWrespectively), to breed with a bird of the opposite phenotypic sex ofnormal chromosome type (chromosally ZZ or ZW respectively), to produceoffspring which are chromosomally homogenetic individuals (all WW).

Third, it ought to be possible to do the same things in othercommercially important avian species. These include, for example,turkeys or pheasant for commercial purposes i.e. human food. They alsoinclude valuable species of birds in the pet trade e.g. parrots, rare orendangered species where one wishes to influence the sex ratio forbreeding purposes. In principle, the mechanism should be applicable toall avian species.

Translating the requirements of the method .of the invention to otheravian species, the temperature shift should be timed to take place at atime of between 2 and 4 days, preferably about 3-3.5 days, before thefirst time at which sexual differentiation can be detectedhistologically, and for a period of time which enables the pulse to becompleted before the time at which sexual differentiation occurs incontrol birds and is not less than 18 hours.

Other ways of creating shifted conditions

The inventor believes that the reason for the improvement in themortality rates following shifts of the incubation temperature relatesto gas and humidity exchange across the egg shell. When one moves fromthe baseline incubation temperature to the shifted temperature and thenback to or near the baseline temperature, there are occasions when theegg is warmer than the incubator, in which case, the egg will lose gasand humidity from the eggshell membrane. Conversely, there are timeswhen the incubator is warmer than the egg, in which case, it will takeup gases and water vapour. This may be important in improving therespiratory exchange of the developing embryo. In nature, whereincubation mortalities are lower, the temperature of egg incubation islikely to oscillate much more than in an artificial incubator. Thoseskilled in the art will therefore be able to find other means ofaltering the ambient environment by use of changes in vapour pressure.

DATA FOR TEMPERATURE PULSE EXPERIMENTS

DP=Day of Incubation after which pulse was applied (24 hour days)

PL=Pulse Length (Hours) PT-Pulse temperature (C)

M=Males F=females D=Dead

                  TABLE 1                                                         ______________________________________                                              PL       PT     #    %     #    %    #    %                             DP    (Hrs)    (°C.)                                                                         M    M     F    F    D    D                             ______________________________________                                        1     24       35     5    50.0  5    50.0 0    0                             2     24       18     18   36.0  27   54.0 5    10.0                          2     24       18     17   34.7  26   53.1 6    12.2                          2     48       18     22   44.9  18   36.7 9    18.4                          2     48       18     26   50.o  19   36.5 7    13.5                          2     24       20     23   44.2  28   53.9 1    1.9                           3     48       20     27   50.9  19   35.9 7    73.2                          3     8        22     26   56.5  17   37.0 3    6.5                           3     8        22     27   50.0  23   42.6 4    7.4                           3     8        22     29   50.9  26   45.6 2    3.5                           3     24       22     25   54.3  19   41.3 2    4.4                           3     24       22     24   52.2  19   41.3 3    6.5                           3     24       22     27   47.3  29   50.9 1    1.8                           3     24       22     35   54.7  25   39.1 4    6.2                           3     24       22     27   48.2  23   41.1 6    10.7                          3     36       22     35   61.4  21   36.8 1    1.8                           3     36       22     26   50.0  25   48.1 1    1.9                           3     36       22     32   51.6  25   40.3 5    8.1                           3     36       22     34   55.7  22   36.1 5    8.2                           3     36       22     33   60.0  20   36.4 2    3.6                           3     48       22     24   54.5  13   29.5 7    16.0                          3     48       22     30   53.6  21   37.5 5    8.9                           3     48       22     20   40.8  21   42.9 8    16.3                          3     48       36     6    28.6  14   66.6 1    4.8                           3     72       36     8    38.1  12   57.1 1    4.8                           3     96       36     14   43.8  17   53.1 1    3.1                           4     48       33     9    40.9  11   50.0 2    9.1                           4     72       33     2    11.1  5    27.8 11   61.1                          4     72       33     2    15.4  5    38.5 6    46.1                          4     96       33     3    13.6  1    4.6  18   81.8                          4     48       34     8    33.3  13   54.2 3    12.5                          4     72       34     6    27.3  10   45.4 6    27.3                          4     96       34     5    20.8  4    16.7 15   62.5                          4     48       35     10   45.5  9    40.9 3    13.6                          4     48       35     11   50.0  11   50.0 0    0                             4     72       35     28   50.0  23   41.1 5    8.9                           4     72       35     6    40.0  9    60.0 0    0                             4     96       35     8    42.1  7    36.8 4    21.1                          4     48       36     7    33.3  6    28.6 8    38.1                          4     72       36     7    30.4  13   56.5 3    13.1                          4     96       36     5    22.7  17   77.3 0    0                             4     96       36     25   47.2  26   49.0 2    3.8                           4     48       37     13   54.2  11   45.8 0    0                             4     72       37     4    19.0  16   76.2 1    4.8                           4     96       37     9    33.3  11   40.7 7    26.0                          5     48       33     10   45.5  11   50.0 1    4.5                           5     72       33     4    19.1  2    9.5  15   71.4                          5     96       33     2    9.1   1    4.5  19   86.4                          5     48       34     10   45.5  7    31.8 5    22.7                          5     72       34     8    32.0  16   64.0 1    4.0                           5     96       34     17   63.0  8    29.6 2    7.4                           5     96       34     23   43.4  22   41.5 8    15.1                          5     48       35     9    39.1  72   52.2 2    8.7                           5     72       35     10   45.5  9    40.9 3    13.6                          5     96       35     14   43.8  13   40.6 5    15.6                          5     48       36     10   26.3  13   34.2 15   39.5                          5     72       36     14   48.3  10   34.5 5    17.2                          5     96       36     10   40.0  7    28.0 8    32.0                          5     48       37     4    17.4  2    8.7  77   73.9                          5     72       37     3    13.0  3    13.0 17   74.0                          5     96       37     4    77.4  5    27.7 14   60.9                          6     48       34     13   31.0  19   45.2 10   23.8                          6     72       34     13   43.3  13   43.3 4    13.4                          6     48       35     9    45.0  10   50.0 I    5.0                           6     72       35     2    33.3  30   50.0 10   16.7                          6     96       35     77   52.4  6    28.6 4    19.0                          6     48       37     1    4.0   2    8.0  22   88.0                          6     72       37     2    8.3   1    4.2  21   87.5                          CONTROL    38     18     45.0  20   50.0 2    5.0                             CONTROL    38     20     52.6  17   44.7 1    2.6                             CONTROL    38     11     45.8  13   54.2 0    0                               CONTROL    38     25     44.6  27   48.2 4    7.2                             CONTROL    38     18     40.9  20   45.5 6    13.6                            CONTROL    38     25     47.2  28   52.8 0    0                               CONTROL    38     21     42.9  25   51.0 3    6.1                             CONTROL    38     23     W.4   32   56.1 2    3.5                             CONTROL    38     26     44.1  30   50.8 3    5.1                             ______________________________________                                    

DATA FOR TEMPERATURE PULSE EXPERIMENTS

DP=Day of Incubation after which pulse was applied (24 hour days)

PL=Pulse Length (Hours) PT=Pulse temperature (C)

M=Males F=females D=Dead

                  TABLE 2                                                         ______________________________________                                              PL       PT     #    %     #    %    #    %                             DP    (Hrs)    (°C.)                                                                         M    M     F    F    D    D                             ______________________________________                                        3     48       20     27   50.9  19   35.9 7    13.2                          3     8        22     26   56.5  17   37.0 3    6.5                           3     24       22     25   54.3  19   41.3 2    4.4                           3     24       22     24   52.2  19   41.3 3    6.5                           3     48       22     20   40.0  16   32.0 14   28.0                          3     48       22     27   50.9  18   34.0 8    15.1                          3     48       22     17   29.8  30   52.6 10   17.6                          3     48       22     19   35.9  28   52.8 6    11.3                          3     48       22     13   37.1  18   51.5 4    11.4                          3     48       22     18   45.0  17   42.5 5    12.5                          3     48       22     15   45.5  15   45.5 3    9.0                           3     48       22     31   43.1  33   45.8 8    11.7                          3     48       22     35   47.9  32   43.9 6    8.2                           3     48       22     24   54.5  13   29.5 7    16.0                          3     48       22     15   36.6  15   36.6 11   26.8                          3     48       25     3    14.3  4    19.0 14   66.7                          3     24       26     9    34.6  13   50.0 4    15.4                          3     48       33     7    31.8  11   50.0 4    18.2                          3     72       33     5    21.7  12   52.2 6    26.1                          3     96       33     7    33.3  1    4.8  13   61.9                          3     48       36     6    28.6  14   66.6 1    4.8                           3     72       36     8    38.1  12   57.7 1    4.8                           3     96       36     14   43.8  17   53.1 1    3.1                           ______________________________________                                    

DATA FOR TEMPERATURE PULSE EXPERIMENTS

    ______________________________________                                        PT = Pulse temperature (C.)                                                                            M = Males                                            PL = Pulse Length (Hours)                                                                              F = females                                          AF = Age of laying flock (Weeks)                                                                       D = Dead                                             EA = Age of egg from time of lay (Hours)                                      Temperature Pulse was applied at day 3 of                                     incubation.                                                                   Number of eggs used in each experiment = 63                                   ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        PT   PL      EA      AF    %     %     %                                      (°C.)                                                                       (Hrs)   (Hrs)   (Wks) M     F     D     M:F                              ______________________________________                                        22    8      18      52    56.5  37.0  6.5   1.53:1                           22    8      36      34    50.0  42.6  7.4   1.17:1                           22    8      30      37    46.0  50.0  4.0   0.92:1                           22    8      36      35    50.9  45.6  3.5   1.12:1                           22   24      18      52    52.2  41.3  6.5   1.26:1                           22   24      36      30    47.3  50.9  1.8   0.93:1                           22   24      36      34    54.7  39.1  6.2   1.40:1                           22   24      36      37    48.2  41.1  70.7  1.17:1                           22   36      36      32    61.4  36.8  1.8   1.67:1                           22   36      36      32    50.0  48.7  1.9   1.04:1                           22   36      36      33    55.7  36.1  8.2   1.54:1                           22   36      30      37    59.2  40.8  0     1.45:1                           22   36      36      33    51.6  40.3  8.1   7.28:1                           22   48      18      52    54.5  29.5  16.0  1.85:1                           22   48      30      36    54.0  38.1  7.9   1.42:1                           22   48      36      30    53.6  37.5  8.9   1.43:1                           22   48      36      30    40.8  42.9  16.3  0.95:1                           CONTROL  36      34      40.4  56.1  3.5   0.72:1                             CONTROL  30      37      43.8  46.9  9.3   0.93:1                             CONTROL  30      37      43.8  53.1  3.1   0.82:1                             CONTROL  36      32      44.1  50.8  5.1   0.87:1                             ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        GENOTYPIC AND PHENOTYPIC SEXING OF THE                                        CHICKS ON THE DAY OF HATCHING                                                 TREATMENT GENOTYPIC SEX   PHENOTYPIC SEX                                      GROUP     (FEATHER COLOUR)                                                                              (VENT SEX)                                          ______________________________________                                        22° C.                                                                            30 male         30 male, 0 female                                  22° C.                                                                            39 female       35 female, 4 male                                  36° C.                                                                           105 male        105 male, 0 female                                  26° C.                                                                           101 female      100 female, 1 male                                  ______________________________________                                    

References

Bennett A. F. and Dawson N. R. (1979). Physiological responses ofembryonic Heermann's gulls to temperature. Physiol. Zool. 52: 413-421.

Deeming D. C. and Ferguson W. R. (1988). Environmental regulation of sexdetermination in reptiles. Phil. Trans. Roy. Soc. London (B) 322: 19-39.

Shubina G. N., Zhmurin L. M. and Vendeneeva V. A. (1972). Effect ofshort-term temperature drop of egg incubation on sex determination ofchicken embryos. TR. Vses. Nauch-Issled. Inst. Fizziol. Biokhim. Pitan.Selskokhoz Zhivotn. 11: 260-268.

Sittman K. (1984) Sex determination in birds: Progeny of nondisjunctioncanaries of Durham (1926), Genetic Research, Cambridge 43: 173-180.

Tone M. et al (1982) Demonstration of W chromosome-specific repetitiveDNA sequences In the domestic fowl Galus Galus domesticus. Chromosoma,86: 551-569: see also

Kodama et al (1987) Chromosoma 96: 18-25; Saitoh et al (1991) Chromosoma101: 32-40; and Saitoh & Mizuno (1992) Chromosoma 101: 474-477.

Tazawa H. and Rahn H. (1986). Tolerance of chick embryos to lowtemperatures in reference to the heart rate. Comp. Biochem. Physiol.85A: 531-534.

I claim:
 1. A method of hatching eggs of avian species which comprisescarrying out at least one cycle of steps (a) to (c) as follows:(a)incubating the eggs in a first, baseline ambient environment which isnormal for hatchery incubation of the eggs of the avian species In theprevailing climatic conditions and then during a sex-sensitive timewindow of embryonic development, (b) altering the ambient environment toshifted conditions for a period of time effective to bias the normalphenotypic sex ratio of the embryos, without significant adverse effecton the average mortality rate, and (c) thereafter restoring theincubation conditions to or towards normal, and allowing the eggs tohatch.
 2. A method according to claim 1, wherein the avian species is achicken.
 3. A method according to claim 2 wherein the shifted conditionscomprise reduced temperature.
 4. A method according to claim 3 whereinthe temperature is reduced when the eggs are at Hamilton-Hamburger stage18 or 19 or at 2-4 days after an incubation which began from 18 to 36hours after the eggs were laid.
 5. A method according to claim 4,wherein the shifted temperature is maintained at about 22° C. for aperiod of from 18 to 42 hours.
 6. A method according to claim 5 whereinthe period of shifted temperature is from 24 to 36 hours.
 7. A methodaccording to claim 4, wherein the shifted temperature is maintained atabout 36° C. for a period of from 18 to 96 hours.
 8. A method accordingto claim 7 wherein the period of shifted temperature is from 24 to 72hours.
 9. A method of producing birds of an avian species which arechromosomally male (ZZ) but phenotypically female or chromosomallyfemale (ZW) but phenotypically male, which comprises hatching eggs tobias the phenotypic sex ratio, by carrying out at least one cycle ofsteps (a) to (c) as follows: (a) incubating the eggs in a first,baseline ambient environment which is normal for hatchery incubation ofthe eggs of the avian species in a prevailing climatic conditions andthen during a sex-sensitive time window of embryonic development, (b)altering the ambient environment to bias the normal phenotypic sex ratioof the embryos, without significant adverse effect on the averagemortality rate, and (c) thereafter restoring the incubation conditionsto or towards normal, and allowing the eggs to hatch, detecting thechromosome type of the hatchlings and selecting hatchlings which arechromosomally male but phenotypically female or vice versa.
 10. A methodaccording to claim 9 which further comprises rearing the hatchlings toan adult age at which they are capable of breeding.
 11. A methodaccording to claim 10 which further comprises allowing thephenotypically female or male birds (chromosomally ZZ or ZHrespectively), to breed with a bird of the opposite phenotypic sex ofnormal chromosome type (chromosomally ZZ or ZW respectively), to produceoffspring which are chromosomally homogenetic individuals (all WW). 12.An incubator for hatching avian eggs adapted to provide at least one ofthe following cycle of conditions: (a) incubating the eggs in a first,baseline ambient environment which is normal for hatchery incubation ofthe eggs of the avian species in the prevailing climatic conditions andthen during a sex-sensitive time window of embryonic development, (b)altering the ambient environment to shifted conditions for a period oftime effective to bias the normal phenotypic sex ratio of the embryos,without significant adverse effect on the average mortality rate, and(c) thereafter restoring the incubation conditions to or towards normal,and allowing the eggs to hatch.
 13. An incubator according to claim 12adapted by the provision of associated programmable control means forvarying the conditions.